In March 1998, the U.S. Fish and Wildlife Services (Service), Region 3 and Region
5 Great Lakes Basin Ecosystem Team (Ecoteam) identified three broad, basin-wide issues on
which to focus its efforts during fiscal years 1998-99. The basin-wide issues include lake
sturgeon restoration and passage, double-crested cormorant, and reauthorization of the
Great Lakes Fish and Wildlife Restoration Act of 1990 (Act). These issues were selected
from among several relatively urgent concerns in the Great Lakes Basin that would benefit
from the combined efforts of Ecoteam members. Addressing these issues meets several
Resource Goals identified by the Ecoteam. Though reauthorization of the Act has been
accomplished, actions on the other two basin-wide issues are continuous and dynamic. Lake
sturgeon restoration and double-crested cormorant issues are in keeping with the Service's
"Fisheries Vision for the Future" and the "Fisheries Action
Plan" which focus on interjurisdictional, native, and depleted species and related
ecosystem impairments.

As a result of the Ecoteams priority issues for the basin, a cross-regional and
cross-program committee was established to identify and address priority action items. The
Lake Sturgeon Committee (Committee) (Appendix A) was formally established in July 1998
with an overall role to provide a mechanism for coordination among the programs, share
information, and seek consistency from a Service perspective. Collaboration of efforts and
expertise from all programs make this an extremely beneficial and efficient process. In
the future, the Committee intends to develop a U.S. Fish and Wildlife Service Great Lakes
Lake Sturgeon Restoration Plan. This Plan will incorporate information collected from the
following priority action items identified by the Committee:

Determine the status of Great Lakes populations for consideration under Endangered
Species Act listing.

Several management agencies throughout the Great Lakes are studying lake
sturgeon populations. Hatchery broodstocks and stocking efforts are utilized as the
primary management tool for restoring or enhancing these populations. Broodstock
production must include maintaining a high level of genetic diversity for environmental
adaptability and fitness (Kincaid 1993). Identifying the genetic makeup of lake sturgeon
populations is crucial to help maximize genetic diversity among broodstocks and to ensure
their integrity is maintained and not contaminated by stocking or transfer events.
Transfer, and the potential recruitment of lake sturgeon from unsuitable strains, can
degrade the fitness of existing populations and potentially limit their ability to achieve
sustainability.

Multiple agencies are collecting different tissue samples to accommodate various analysis
methodologies. An analysis includes identifying genetic markers that detect polymorphisms
within and among individuals. A sufficient sample size must be acquired to determine if
enough variation exists within that particular stock. If so, differentiation among stocks
can be detected. The Committee needs to address the compatibility of the techniques and
sampling regimes (i.e. cooperation) currently being used by various agencies.

ObjectiveThe objective of this report is to compile information on existing genetic samples and
programs conducted by various natural resource agencies to determine compatibility and
identify potential standardization methods.

Below are descriptions of the activities and composition of genetics samples within
each agency associated with Great Lakes Basin lake sturgeon work through May 1999. See
Table 1 for a summary of key components. The information has been compiled from documents,
as well as correspondences between geneticists and biologists.

The Ashland Fishery Resources Office (FRO) is a member of the Lake Sturgeon Sub-Committee
of the Lake Superior Committee. Lake sturgeon genetic samples are obtained from several
cooperators/partners including the Marquette Biological Station, Wisconsin and Minnesota
Departments of Natural Resources (DNR), and Michigan Technological University (MTU). In
1995 and 1997, the Ashland FRO obtained 31 muscletissue samples (Porter et al.
1997) and 64 fin samples, respectively from the Bad River.

Dr. Paul Fuerst at Ohio State University (OSU) (Appendix B) has received all samples
obtained by the Ashland FRO. Dr. Fuerst is conducting genetic analyses with Dr. Ted
Cavender of the Ohio Museum of Biological Diversity (Appendix B) and several graduate
students. Their objectives include determining the genetic structure of natural Great
Lakes lake sturgeon populations, providing an interpretation of this information to assist
state and federal agencies, and using museum specimens to compare current genetic
diversity to historical levels of genetic diversity. The current methods include obtaining
fin, muscle, or blood tissue to perform biochemical (allozymes) and molecular (RAPDs,
microsatellite loci, mitochondrial DNA sequences, unique variable nuclear genes) analyses.
Population differentiation is determined by looking at allele frequency changes
(allozymes, microsatellite, and mtDNA), band sharing (VNTR and RAPD loci), and DNA
sequence divergence (mtDNA and unique variable sequences). The full analysis involves
examining at least 16 populations. The document The Genetic Status of Lake Sturgeon in
Lake Erie and other Populations from the Laurentian Great Lakes(Porter et
al. 1995) is currently available which describes findings from some of their research.
Future objectives of the project are to compare differences between sex, drainages within
a lake, sites within a drainage, breeders and non-breeders, and between successive years
of breeders. No date of completion has been identified for future work.

In 1995 and 1996, MTU provided 45 muscle samples from the Sturgeon River to Dr. Fuerst
(Porter et al. 1995). Minnesota DNR and Wisconsin DNR forwarded 30 muscle, blood,
fin, eye, and liver samples from St. Louis Bay lake sturgeon and 30 muscle, blood, and fin
samples from Chequamegon Bay to Dr. David Philipp and Dr. Bob Fields at the Illinois
Natural History Survey (Appendix B) (Fields and Philipp Unpublished). Samples from the
Lake Michigan Basin (see below) and Mississippi River Basin (Flambeau, Snake Rivers)
accompanied the Lake Superior Basin samples to Dr. Philipp. Dr. Philipp and Dr. Fields
used a PCR-RFLP analysis of mtDNA and protein electrophoresis in their methods (Fields and
Philipp Unpublished). A publication of the results is in progress.

In 1994 and 1996, the Michigan DNR, Marquette Field Station collected 25 muscle and blood
samples and 30 fin tissue samples, respectively from the Menominee River. These samples
were forwarded to Dr. Fuerst (Porter et al. 1995).

LaCrosse Fishery Resources Office - Ann Runstrom

The LaCrosse FRO obtained sixlake sturgeon fin tissue samples from the Mississippi
River. Although outside of the Great Lakes Basin, there is fish movement between Lake
Michigan and the Mississippi River via the Illinois (Chicago) Waterway. Currently, the
LaCrosse FRO holds these samples for future analysis, with no geneticist identified to
conduct the work. In addition, the LaCrosse FRO initiated a cooperative project with Dr.
Scott Cooper of the University of Wisconsin at LaCrosse (Appendix B) to investigate
species differentiation using genetic material from eggs. Ths information was critical due
to the inability to identify species eggs in the wild based only on appearance.
Primarily, shovelnose sturgeon and paddlefish from the Mississippi drainage were studied;
however, some work was conducted with pallid and lake sturgeons. The lake sturgeon eggs
from the Wolf River population were collected by the Wild Rose Fish Hatchery in Wisconsin.
Dr. Cooper used a polymerase chain reaction (PCR) -based test to amplify a portion of the
cytochrome-B gene from the mtDNA of a single egg. The presence of distinct HincII restriction
sites (markers) in the PCR product allowed rapid determination of shovelnose sturgeon,
pallid sturgeon, and paddlefish. Distinct restrictions sites did not show up in the lake
sturgeon eggs, which separated this species from the other three. The procedure and
reagents have been forwarded to the USFWS forensics lab in Ashland, Oregon to apply
towards the illegal trade of caviar (see Law Enforcement below).

The Alpena FRO is the coordinator of the Central Great Lakes Bi-National Lake Sturgeon
Group. Lake sturgeon genetic samples have been obtained from several cooperators/partners
including Michigan and Ohio DNRs and Ontario Ministry of Natural Resource offices (OMNR).
The Alpena FRO currently has 20 (or more) lake sturgeon fin tissue samples from the
Detroit River and approximately 10-15 from Saginaw Bay, Lake Huron fish. All samples were
collected in fall 1998. Samples will be forwarded to Dr. Paul Fuerst (OSU), and to Dr. Kim
Scribner (MSU) in cooperation with Michigan DNR.

In 1993-1996, Michigan DNR provided approximately 40 muscle, fin, and blood samples from
Lake St. Clair lake sturgeon to Dr. Fuerst as well as 30 fin tissues from Black Lake in
1997 (Porter et al. 1997). In 1998, they obtained 95 fin samples from the St. Clair
River and 140 from Lake St. Clair. These were forwarded to Dr. Scribner who will utilize
microsatellite and mtDNA analysis techniques. The mtDNA work will begin with direct DNA
sequencing, possibly followed by PCR-based tests and restriction sites.

The Lower Great Lakes FRO has obtained 29 lake sturgeon fin tissues from several agencies.
Tissue samples were collected from the following water bodies; eastern Lake Erie (6),
upper Niagara River (2), lower Niagara River (11), and Lake Ontario (10). Samples have
been collected by the Lower Great Lakes FRO since 1994, with the majority being obtained
in 1996 and 1998. Samples from 1994 to 1996 were forwarded directly to Dr. Fuerst, and to
Dr. Bernie May (University of California at Davis) via Harold Kincaid at the USGS- BRD
laboratory in Wellsboro, PA (Appendix B). The 1997 and 1998 samples were only forwarded to
Dr. May. In addition, the 1998 samples were sent to Dr. Tim King at the USGS-BRD, Leetown
Science Center in West Virginia (LSC) (Appendix B).

The objectives of Dr. May and Dr. Kincaids work includes: (1) characterize stocks
with genetic markers, (2) develop standardized DNA analysis for sturgeon broodstocks (see
Discussion below), (3) measure genetic diversity in sturgeon species, (4) provide a
centralized database for genetic information, and (5) assist fisheries management agencies
develop genetic management programs. Methods involve using fin tissue to develop primers
for microsatellite loci to characterize within- and between-population variation. Dr. May
and his colleagues have published their lake sturgeon markers, which can be used by other
geneticists (May et al. 1997). A study to identify the inheritance mechanisms of
microsatellite loci in lake sturgeon has been performed by the investigators, with a
manuscript being prepared for publication in 1999. Also, final results of the
microsatellite screening and population differentiation are expected to be completed in
1999.

The Leetown Science Center has primarily been working with Atlantic sturgeon; however,
more recently has performed lake sturgeon genetic analysis. The LSC has developed markers
for Atlantic sturgeon and have tested them on our lake sturgeon samples. The Atlantic
sturgeon markers are available at Genbank, a clearinghouse through the National Library of
Medicine and a publication identifying the markers is currently in peer review. The
Atlantic sturgeon markers and Dr. Mays lake sturgeon markers can be utilized by the
LSC to evaluate future samples. Methods include using fin tissue and the microsatellite
loci primers to determine genetic variation.

Between 1994-1996, the New York State Department of Environmental Conservation provided
Dr. Fuerst with 15 blood samples from St. Lawrence River lake sturgeon below the Robert
Moses Power Project and 7 blood samples from Grasse River lake sturgeon. Barbel samples
collected in 1997 and 1998 have been forwarded to Dr. Rejean Fortin (Appendix B) in
cooperation with lower St. Lawrence River studies (see sections below).

In 1998, the Lake Champlain Fish and Wildlife Resources Office (LCFWRO) obtained three fin
tissue samples from the LeMoile River, tributary to Lake Champlain. The Vermont Department
of Fish and Wildlife currently has the samples, which likely will be forwarded to Dr.
Louis Bernatchez at Laval University in Quebec via Rejean Fortin (Appendix B). Colleagues
of Dr. Fortin have looked at mtDNA variation in St. Lawrence River lake sturgeon
(Guénette et al. 1993); however, all future work is expected to be conducted by
Dr. Bernatchez who will use the microsatellite loci developed by Dr. May (lake sturgeon)
and Dr. King (Atlantic sturgeon). Both Fortin and Bernatchez use barbel samples.

The forensics laboratory has currently performed species identification on 17 sturgeon
species using mtDNA from the eggs. Regarding lake sturgeon, most work has been performed
on meat samples rather than caviar and they have no difficulty identifying them. In the
future, the lab will use microsatellite techniques on the North American sturgeon species.
This will help identify populations within a species.

This report provides an inventory of lake sturgeon genetic samples in the Great Lakes
Basin as well as a brief overview of analysis techniques being used. Based on the
information reported by participating agencies, it is apparent that enhanced interagency
coordination is necessary to guide future research on this topic. Standardization of lake
sturgeon genetics work is possible; however, the issues presented below must be addressed
in the near future.

The best collection, fixing, and preserving method should be identified and standardized
among agencies to obtain clean and functional samples. Several tissue types have been
obtained for genetic analysis (Table 1). Fortunately, tissue type does not cause a
conflict in comparing analyses. However, different collection, fixing, and preserving
methods are being used, which requires extensive care in the field and laboratory to avoid
contamination of the sample. A contaminated sample can result in an inaccurate analysis,
leading to false comparisons. In 1999, a recommendation was made to all agencies to
continue collecting and preserving tissue samples as instructed by their respective
geneticist; however, to keep their samples until enhanced coordination could occur.

The results of mitochondrial DNA (mtDNA) and microsatellite analyses are not directly
comparable. There are both advantages and disadvantages of using each technique for
genetic stock identification; however, Ferguson and Danzmann (1998) suggest both genome
analyses should be surveyed extensively to obtain the best representation of a population.
The two results can be combined rather than compared.

If the same analysis method is being used between geneticists, discrepancies can still
occur when different markers, which are developed by the individual geneticist, are used.
In brief, mtDNA analysis is usually performed by direct base pair sequencing or by using
restriction enzymes (markers), which recognizes and cuts the DNA at a particular base pair
sequence (i.e. allele). Microsatellite analysis looks for a specific base pair sequence
and quantifies the number of repeats of that sequence. Again, comparing separate analyses
becomes difficult because the markers identify specific alleles that can differ depending
on the base pair sequences being utilized, and it is uncertain if all geneticists are
using the same base pair sequence (e.g. allelic identification). Often an automated
sequencer is used for precision; however, two sequencers can also differ in allele
identification. Phospho-imaging is another technique that can be a source of variation in
allelic designations.

A coordinated effort to identify lake sturgeon genetic needs must occur among state,
federal, and provincial resource managers and biologists in the Great Lakes Basin. These
needs must be articulated to research geneticists, who in turn can provide practical
information needed to manage lake sturgeon populations. In general, to guide restoration
and enhancement efforts in the Great Lakes, resource managers need answers regarding
population differentiation. Regardless of using mtDNA, microsatellite, or both,
information from the available markers has not been fully utilized to assess population
structure. Also, additional markers may be needed. A sturgeon genetic marker library is
being developed, using microsatellite markers, to compile available sturgeon markers and
determine their utility to assess population structure. Researchers contributing to this
effort include Dr. Bob Sheehan at Southern Illinois University (shovelnose sturgeon), Dr.
May (lake sturgeon), and Dr. King (Atlantic sturgeon). Potentially, this is a positive
step toward standardizing analyses due to a large number of markers being developed. In
addition, it will allow multiple geneticists to use the same identified alleles, and
likely provide enough information for definitive population differentiation.

The Lower Great Lakes Fishery Resources Office is in the process of coordinating a
genetics workshop, including resource biologists, managers, and geneticists, to discuss
standardization of Great Lakes lake sturgeon genetics activities. The expected outcomes
from this workshop include: (1) sharing current genetics capabilities/technology with all
participants, (2) identifying information and research needs, (3) identifying the best
collection and analysis methods, (4) establishing a network of communication between
agencies and geneticists, and (5) identifying funding sources to conduct the necessary
research.

The Great Lakes Basin Ecosystem Team, Lake Sturgeon Committee would like to thank all
of the participating agencies and contact personnel who provided information on their lake
sturgeon genetics activities. Also, a special thanks is given to the geneticists for their
time and patience during numerous conversations to ensure accuracy in the information
presented. Also, the author thanks all reviewers for their time critiquing previous
drafts.

Ferguson, M.M. and R.G. Danzmann. 1998. Role of genetic markers in fisheries and
aquaculture: useful tools or stamp collecting. Canadian Journal of Fisheries and Aquatic
Science 55:1553-1563.

Guénette, S., R. Fortin, and E. Rassart. 1993. Mitochondrial DNA variation in lake
sturgeon (Acipenser fulvescens) from the St. Lawrence River and James Bay drainage
basins in Quebec, Canada. Canadian Journal of Fisheries and Aquatic Sciences
50(3):659-664.

Kincaid, H.L. 1993. Breeding plan to preserve the genetic variability of the Kootenai
River white sturgeon. Bonneville Power Adminstration. 22pp.